Identification of a new methylation site in the Sept9 promoter region for the diagnosis of hepatocellular carcinoma

Background. Over 600,000 people die from hepatocellular carcinoma (HCC) each year worldwide. The disease is often detected at advanced stages and in many cases is not curable. Early diagnostic and monitoring of HCC recurrences remains a substantial problem in clinical oncology. That determines the need for a search for highly sensitive and specific biomarkers for the non-invasive of HCC diagnostics. The objective of the study. Identification of the hypermethylated locus in the promoter region of the septin 9 (Sept9) gene based on the annotated methylomes from the public databases. Experimental validation of methylation on a pilot panel of paired clinical samples of patients with HCC, as well as tissue samples from patients with benign liver tumors and lymphocytes from healthy donors. Materials and methods. To analyze the methyl data, samples of HCC from TCGA, hepatocellular adenoma from GEO (Gene Expression Omnibus) depository, peripheral blood cells and tissues of healthy donors from Methbank were used. Experimental validation of methylation levels of the identified site was carried out on a pilot panel of clinical samples by bisulphite pyrosequencing using PyroMark Q24.Results. Based on the analysis of methylome data, we selected cg20275528 site, which is characterized by high level of methylation in HCC tissues and minimal levels of methylation in non-tumor liver tissue, hepatocellular adenoma and peripheral blood of healthy donors. Experimental testing on a pilot panel of clinical specimens showed that the level of marker site methylation in HCC (42 % median) is significantly higher than in non-tumor liver tissues (3 % median) and benign neoplasms (1.5 % median) and exceeds the threshold value in HCC compared to paired samples of adjacent non-tumor liver tissue in 20 out of 30 studied cases (66.6 %). The general possibility for cg20275528 methylation detection in circulating DNA of plasma in HCC patients was shown.Conclusion. The obtained results indicate that the approach to the detection and experimental verification of diagnostically significant markers developed and tested in this study can be used to identify new differentially methylated sites and to establish new approaches for non-invasive HCC diagnosis.

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Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids

Scientific Reports ◽

10.1038/s41598-021-86910-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jingjing Zhang ◽

Luong T. H. Nguyen ◽

Richard Hickey ◽

Nicole Walters ◽

Xinyu Wang ◽

...

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Metastatic Breast ◽

Clinical Samples ◽

Clinical Use ◽

Immunomagnetic Beads ◽

Liquid Biopsies ◽

Cell Culture Media ◽

Non Invasive ◽

Healthy Donors

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5–100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

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Immunomagnetic Sequential Ultrafiltration (iSUF) Platform for Enrichment and Purification of Extracellular Vesicles from Biofluids

10.1101/2020.05.13.089573 ◽

2020 ◽

Cited By ~ 2

Author(s):

Jingjing Zhang ◽

Luong TH Nguyen ◽

Richard Hickey ◽

Nicole Walters ◽

Xinyu Wang ◽

...

Keyword(s):

Extracellular Vesicles ◽

Culture Media ◽

Metastatic Breast ◽

Clinical Samples ◽

Immunomagnetic Beads ◽

Liquid Biopsies ◽

Cell Culture Media ◽

Non Invasive ◽

Healthy Donors ◽

Small Cohort

AbstractExtracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs (nonspecifically and specifically) in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5 mL to 100 mL) can be significantly enriched (up to 1000 times), with up to 99 % removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads specific to a target subpopulation of EVs. Serum from a small cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

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Qualitative elastography and arfi technology in the diagnosis of liver tumors

Journal of New Medical Technologies eJournal ◽

10.12737/6670 ◽

2014 ◽

Vol 8 (1) ◽

pp. 0-0

Author(s):

Гудилина ◽

E. Gudilina ◽

Вишленкова ◽

E. Vishlenkova ◽

Лепэдату ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Positive Predictive Value ◽

Predictive Value ◽

Liver Tumors ◽

Malignant Tumors ◽

Hepatocellular Adenoma ◽

Liver Parenchyma ◽

Benign Tumors ◽

Area Of Interest ◽

Arfi Elastography

The aim of this study was to evaluate the stiffness of liver tumors using compression and ARFI elastography. Compression and ARFI elastography was performed to study the color display rigidity in the foci of the liver in 69 and 81 patients, respectively. Area of interest was placed in the education and on the border with the surrounding liver parenchyma, to assess the visual difference tissue stiffness. Patients were divided by diagnosis: hepatocellular carcinoma – 36, cholangiocarcinoma – 6 metastases – 35, benign – 4 cases. Benign tumors included two focal nodular hyperplasia, hepatocellular adenoma and one cavernous hemangioma a large size. When compression elastography increased stiffness formations compared to the surrounding liver parenchyma was observed: in benign – 100%, hepatocellular carcinoma – in 71.9%, cholangiocarcinoma – 80%, metastases – in 72.4%. When ARFI elastography increased stiffness observed: in benign – 100%, hepatocellular carcinoma – 58.3%, cholangiocarcinoma – 100%, metastases – in 85.7%. Qualitative elastography improves the definition of clear boundaries neoplasm infiltration beyond the tumor, but can not differentiate between malignant and benign tumors. Sensitivity, accuracy, positive predictive value of the compressive elastography were as follows: 73, 69, 94%, and at ARFI elastography – 74, 70, 93%, respectively. Both techniques are qualitative elastography complement each other, with the specification of the internal structure of foci and their joint application sensitivity, accuracy, positive predictive value in the diagnosis of malignant tumors accounted for 83, 79, 95%, respectively. ARFI elastography in conjunction with compression elastography improves the visualization of malignant tumors of the liver and can be used as an additional diagnostic tool in oncohepatology.

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Salivary miRNAs as non-invasive biomarkers of hepatocellular carcinoma: a pilot study

PeerJ ◽

10.7717/peerj.12715 ◽

2022 ◽

Vol 10 ◽

pp. e12715

Author(s):

Arshiya Mariam ◽

Galen Miller-Atkins ◽

Amika Moro ◽

Alejandro I. Rodarte ◽

Shirin Siddiqi ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Disease ◽

Chronic Liver Disease ◽

Liver Tissue ◽

Chronic Liver ◽

Detection Methods ◽

Support Vector ◽

Small Rna Sequencing ◽

Significant Differential Expression ◽

Non Invasive

Background Improved detection of hepatocellular carcinoma (HCC) is needed, as current detection methods, such as alpha fetoprotein (AFP) and ultrasound, suffer from poor sensitivity. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate many cellular functions and impact cancer development and progression. Notably, miRNAs are detectable in saliva and have shown potential as non-invasive biomarkers for a number of cancers including breast, oral, and lung cancers. Here, we present, to our knowledge, the first report of salivary miRNAs in HCC and compare these findings to patients with cirrhosis, a high-risk cohort for HCC. Methods We performed small RNA sequencing in 20 patients with HCC and 19 with cirrhosis. Eleven patients with HCC had chronic liver disease, and analyses were performed with these samples combined and stratified by the presence of chronic liver disease. P values were adjusted for multiple comparisons using a false discovery rate (FDR) approach and miRNA with FDR P < 0.05 were considered statistically significant. Differential expression of salivary miRNAs was compared to a previously published report of miRNAs in liver tissue of patients with HCC vs cirrhosis. Support vector machines and leave-one-out cross-validation were performed to determine if salivary miRNAs have predictive potential for detecting HCC. Results A total of 4,565 precursor and mature miRNAs were detected in saliva and 365 were significantly different between those with HCC compared to cirrhosis (FDR P < 0.05). Interestingly, 283 of these miRNAs were significantly downregulated in patients with HCC. Machine-learning identified a combination of 10 miRNAs and covariates that accurately classified patients with HCC (AUC = 0.87). In addition, we identified three miRNAs that were differentially expressed in HCC saliva samples and in a previously published study of miRNAs in HCC tissue compared to cirrhotic liver tissue. Conclusions This study demonstrates, for the first time, that miRNAs relevant to HCC are detectable in saliva, that salivary miRNA signatures show potential to be highly sensitive and specific non-invasive biomarkers of HCC, and that additional studies utilizing larger cohorts are needed.

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Distinct Changes of BTLA and HVEM Expressions in Circulating CD4+ and CD8+ T Cells in Hepatocellular Carcinoma Patients

Journal of Immunology Research ◽

10.1155/2018/4561571 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Jiayu Liu ◽

Jiaqian Li ◽

Min He ◽

Geng-Lin Zhang ◽

Qiyi Zhao

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Critical Role ◽

T Cell Subsets ◽

Strong Positive Correlation ◽

T Cell Suppression ◽

Healthy Donors ◽

Cell Suppression

BTLA/HVEM (B and T lymphocyte attenuator/herpes virus entry mediator) pathways play a critical role in T cell suppression in tumor. However, its dynamic changes in different T cell subsets in peripheral blood and their clinical significance are largely unclear in cancer patients. In the current study, we showed distinct changes of BTLA and HVEM expressions on peripheral blood CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC); BTLA expression were significantly upregulated on circulating CD4+ but not CD8+ T cells. In sharp contrast, the levels of HVEM expression were significantly downregulated on circulating CD8+ but not CD4+ T cells. A strong positive correlation between BTLA expression on circulating CD4+ T cells and BTLA expression on autologous CD8+ counterparts was observed in healthy donors but absent in HCC patients. More importantly, we found that blockade of the BTLA/HVEM pathway increased IFN-γ production in both circulating CD4+ and CD8+ T cells. Collectively, our data suggested that the BTLA/HVEM pathway contributes to peripheral T cell suppression in HCC patients, and BTLA/HVEM may serve as attractive targets for HCC immunotherapy.

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